The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the "nonribosomal code" of A-domains.
Bibliographical noteFunding Information:
This work was supported by a lab startup grant from the University of Chicago (to J.Y.) and a grant from the National Institutes of Health (AI070219 to C.C.A.). We thank Professor K. Dane Wittrup of the Massachusetts Institute of Technology for providing the pCTCON2 plasmid and yeast strain EBY100. We also thank S. Annie Gai and Tiffany F. Chen of the Wittrup Group and Satoe Takahashi, Stephen Kron, Akiko Koide, and Shohei Koide of the University of Chicago for helpful discussions.